Polypeptide cartilage-inducing factors found in bone

ABSTRACT

Two proteins that are found in bone and that have in vivo chondrogenic/osteogenic activity in combination with a co-factor are described. Both proteins also were active in combination with EGF in the in vitro TGF-β assay. Each has a molecular weight of approximately 26,000 daltons by SDS-PAGE. Each is reduced to a single polypeptide indicating that the proteins are probably homodimers. One has an N-terminal sequence identical to that of human placenta-derived TGF-β whereas the other has an N-terminal sequence that is different from that of TGF-β derived from human placenta. The two proteins may be purified to homogeneity using RP-HPLC or acetic acid-urea gel electrophoresis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a .Iadd.continuation of application Ser. No.07/664,766, filed Mar. 5, 1991 abandoned which is a RE of 07/204,173,filed Jun. 8, 1988, now U.S. Pat. No. 4,843,063, which is a.Iaddend.continuation of copending U.S. Ser. No. 129,864, filed Dec. 10,1987, now U.S. Pat. No. 4,774,322, issued Sept. 27, 1988, which is acontinuation of U.S. Ser. No. 767,144, filed Aug. 19, 1985, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 630,938,filed July 16, 1984, now abandoned.

DESCRIPTION

1. Technical Field

The present invention relates to protein chemistry. More particularly,it relates to polypeptides that are involved in cartilage induction invivo. These polypeptides are sometimes referred to as cartilage-inducingfactors (CIFs) herein.

2. Background

Parent U.S. patent application Ser. No. 630,938 describes two proteinsthat were isolated from bovine bone. These proteins exhibited in vitrochondrogenic activity. Further characterization and testing of these twoproteins revealed that one or them may be the same polypeptide as ahuman placenta-derived polypeptide called "beta type transforming growthfactor" (TGF-β) and described in International patent applicationPCT/US83/01460 published Mar. 29, 1984 under No. WO 84/01106 and EPA No.84450016.5 published Dec. 19, 1984 under No. 0128849. The other has adifferent partial amino acid sequence than that reported for the humanplacenta-derived TGF-β, but is active in the same anchorage-independentcell growth assay as was used to characterize the biological activity ofTGF-β.

There have been prior attempts to identify and isolate factors in bonethat are involved in stimulating bone growth. U.S. Pat. No. 4,294,753 toUrist reports the preparation of a crude "bone morphogenic protein" byextracting demineralized bone with urea or guanidine hydrochloride andreprecipitating protein from the extract. Urist subsequently reported(Urist. M. R., Clin Orthop Rel Res (1982) 162:219) that ion exchangepurification of this crude protein mixture yielded an activity that wasunadsorbed to carboxymethylcellulose (CMC) at pH 4.8. Urist's mostrecent reports (Science (1983) 220:680-685 and Proc Natl Acad Science(USA) (1984) 81:371-375) describe BMPs having molecular weights of17,500 and 18,500 daltons.

U.S. Pat. No. 4,434,094 reports the partial purification of a bonegeneration-stimulating, bone-derived protein factor by extraction withchaotropic agents, fractionation on anion and cation exchange columns,and recovery of the activity from a fraction adsorbed to CMC at pH 4.8.This new protein fraction was termed "osteogenic factor" (OF) and wascharacterized as having a molecular weight below about 30,000 daltonsand as tracking the purification process described. The proteins of thecurrent invention were purified to homogeneity using a purificationprocedure that is similar in part to that disclosed in U.S. Pat. No.4,434,094.

DISCLOSURE OF THE INVENTION

The invention provides two CIFs found in mammalian bone that:

(1) are co-factors for inducing cartilage formation in vivo.

(2) promote connective tissue deposition in vivo in the absence of anyadded activating agent or co-factor, and

(3) are active in the anchorage-independent cell growth assay used tocharacterize TGF-β (this assay is sometimes called the "TGF-β assay"herein and is described in Methods for Preparation of Media,Supplements, and Substrata for Serum-Free Animal Cell Culture (1984) pp181-194, Alan R. Liss, Inc.). These two polypeptides are called CIF-Aand CIF-B, respectively, herein.

The invention further provides a process for obtaining CIF-A and CIF-Bin substantially pure form from bone. In this regard the presence inbone of proteins having activity in the TGF-β assay has not beenreported previously. CIF-A has a partial (30 amino acids) N-terminalamino acid sequence reported for human placenta-derived TGF-β. Butlacking total sequencing of both molecule, it is not known whether theyare the same polypeptide. CIF-B has a different partial (30 amino acids)N-terminal amino acid sequence. The invention further providescompositions and methods for promoting proliferation of cells and forpromoting connective tissue deposition using the CIFs.

Accordingly, one aspect of the invention is a polypeptide that: (a) isfound in mammalian bone, (b) is a co-factor for inducing cartilageformation, (e) is active in the TGF-β assay, (d) is a dimer ofapproximately 26,000 daltons as determined by SDS-PAGE, and (e) issubstantially pure.

The process for isolating the CIFs from bone involves;

(a) extracting demineralized bone (DMB) with a chaotropic (dissociative)extractant that solubilizes nonfibrous proteins;

(b) subjecting the extract from step (a) to gel filtration to recover afraction containing proteins of molecular weight 10,000-40,000 daltons;

(c) adsorbing the fraction from step (b) onto a carboxymethyl cellulosecation exchanger at approximately pH 4.5-5.5 under denaturingconditions;

(d) eluting the adsorbed fraction from the cation exchanger with asodium chloride gradient;

(e) subjecting the portion of the eluate from step (d) that elutes atapproximately 150 to 250 mM sodium chloride to reverse phase highperformance liquid chromatography (RP-HPLC) or a nondenaturing gelelcotrophoresis; and

(f) recovering the CIFs from the RP-HPLC or gel electrophoresis.

The composition for promoting proliferation of normal animal cellscomprises;

(a) at least one of the above-described polypeptides;

(b) a TGF-β activating agent; and

(c) a pharmaceutically acceptable carrier, the polypeptide andactivating agent being present in amounts sufficient to promote cellproliferation.

The method of promoting normal cell proliferation comprisesadministering an effective amount of the above-described composition toa patient.

The composition for promoting connective tissue deposition comprises:

(a) an effective amount of one or both of the above-describedpolypeptides substantially free of co-factor or activating agent, and

(b) a pharmaceutically acceptable carrier.

The method of promoting connective tissue deposition comprises placingthe above-described composition at a predetermined site within the bodyof a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a graph of the optical densities (absorbances) (280 nm) and invitro chondrogenic activities of the gel filtration fractions of theexample (section C), infra;

FIG. 2 is a graph of the optical densities (280 nm) of eluate fractionsfrom the preparative ion exchange chromatography of the example (sectionD), infra;

FIG. 3 is a chromatogram and autoradiograph of the SDS-PAGE analysis ofthe RP-HPLC resolved CM-1 fraction described in section E of theexample, infra.

FIG. 4 is a graph of the UV absorbance and electrophoretic profiles ofpeaks A and B of the preparative RP-HPLC of the example (section E),infra;

FIG. 5 is a graph of the results of the enzyme-linked immunosorbentassays (ELISAs) for in vitro chondrogenic activity of the proteins ofpeaks A and B from the RP-HPLC of the example (section E), infra;

FIG. 6 is a graph of the results of the ELISAs of the acid-urea gelelectrophoresis fractions (section F) of the example, infra; and

FIG. 7 is a graph of the results of the TGF-β assays described insection I of the example, infra.

MODES OF CARRYING OUT THE INVENTION

The polypeptides of the invention were isolated from bovine bone. Thepolypeptides have been only partially sequenced at this time. in view ofthis and since the complete amino acid sequence of TGF-β has not beenreported, the primary structure relationships between the invention CIFsand TGF-βs are not known completely. However, based on comparisons ofpartial sequences of N-terminii, molecular weight analyses, apparentsecondary structures, and biological activity, CIF-A may be identical orsubstantially homologous to TGF-β derived from human placenta. CIF-A is,however, more prevalent in bone than TGF-β is in platelets, placenta, orkidney. Thus, if the two are the same molecule, it appears more likelythat the molecule acts as a chondrogenic/osteogenic agent in its nativesetting rather than as a transforming growth factor.

The CIFs have in vitro chondrogenic activity by themselves, in vivochondrogenic activity in combination with certain chondrogenicco-factors, in vivo connective tissue deposition activity by themselves,and in vitro TGF-β activity in combination with epidermal growth factor(EGF). In view of their chondrogenic activity and the mode ofendochondral bone formation, they are also expected to have in vivoosteogenic activity in combination with chondrogenic co-factors.

In view of the showings that bone inductive proteins from human, monkey,bovine and rat are nonspecies-specific in their abilities to produceendochondral bone in xenogeneic implants (Sampath, T. K., et al, ProcNatl Acad Sci (USA) (1983) 80:6591) and that human platelet/humanplacenta/bovine kidney-derived TGF-β is nonspecies-specific betweenrodents, cattle, and humans, it is believed that the polypeptides ofthis invention have been highly conserved among mammalian species (i.e.,polypeptides from different mammalian species have amino acid sequencesthat vary, if at all, in one or more amino acid residue additions,deletions, or substitutions that do not affect that nonspecies-specificactivity of the molecule adversely). In this regard the term"substantially equivalent" as used to describe a polypeptide is intendedto mean polypeptides, whether native or synthetic and regardless ofspecies or derivation, that have the same amino acid sequence as thereferenced polypeptide, and polypeptides of substantially homologous(i.e., at least 90% identity in amino acid sequence) but different aminoacid sequence, which difference(s) does not affect nonspecies-specificactivity adversely. Accordingly, the polypeptides of this invention maybe derived from or found in cells or tissue of diverse animal origin orsynthesized by recombinant DNA technology. Porcine or bovine long boneare preferred native sources of the polypeptides because of the readyavailability of such bone and the abundance of the polypeptides in bone.

A variety of initial preparation procedures are possible when bone isused as a source, but basically the bone is first cleaned usingmechanical or abrasive techniques, fragmented, and further washed with,for example, dilute aqueous acid preferably at low temperature, and thendefatted by extraction with a lipophilic solvent such as ether or ethylacetate. The bone is then demineralized by removal of the calciumphosphates in their various forms, usually by extraction with strongeracid. These techniques are understood in the art, and are disclosed, forexample, in U.S. Pat. No. 4,434,094. The resulting preparation, ademineralized bone, is the starting material for the preparation of thepolypeptides of the invention.

The initial extraction is designed to remove the non-fibrous (e.g.,non-collagenous) proteins from the demineralized bone. This can be donewith the use of chaotropic agents such as quanidine hydrochloride (atleast about 4 molar), urea (8 molar) plus salt, or sodium dodecylsulfate(at least about 1% by volume) or such other chaotropic agents as areknown in the art (Termine, et al, J Biol Chem (1980) 255:9760-9772; andSajera and Hascall, J Biol Chem (1969) 244:77-87 and 2384-2396). Theextraction is preferably carried out at reduced temperatures in thepresence of a protease inhibitor to reduce the likelihood of digestionor denaturation of the extracted protein. Examples of proteaseinhibitors that may be included are phenylmethylsulfonylfluoride (PMSF)sodium azide, N-ethyl maleimide (NEM), benzamidine, and 6-aminohexanoicacid. The pH of the medium depends upon the extractant used. The processof extraction generally takes on the order of about 4 hr to one day.

After extraction, the extractant may be removed by suitable means suchas dialysis against water, preceded by concentration of ultrafiltrationif desired. Salts can also be removed by controlled electrophoresis, orby molecular sieving, or by any other means known in the art. It is alsopreferred to maintain a low temperature during this process so as tominimize denaturation of the proteins. Alternatively, the extractantneed not be removed, but rather the solution need only be concentrated,for example, by ultrafiltration.

The extract, dissolved or redissolved in chaotropic agent, is subjectedto gel filtration to obtain fractions of molecular weight below about40,000 daltons, thus resulting in a major enhancement of purity. Gelsizing is done using standard techniques, preferably on a Sephacrylcolumn at room (10°-25° C.) temperature. The low molecular weightfraction is then subjected to ion exchange chromatography usingcarboxymethyl cellulose (CMC) at approximately pH 4.5-5.2, preferablyabout 4.8, in the presence of a nonionic chaotropic agent such as urea.Other cation exchangers may be used, including those derived frompolyacrylamide and cross-linked dextran; however cellulosic cationexchangers are preferred. Of course, as in any ion exchange procedure,the solution must be freed of competing ions before application to thecolumn, and is eluted in an increasing salt concentration gradient as isunderstood in the art. The fraction eluting from CMC at about 150 to 250mM NaCl contains the CIFs.

The eluate fraction from the cation exchange chromatography is thensubjected to RP-HPLC or a non-denaturing gel electrophoresis for finalpurification. Standard RP-HPLC techniques and gel electrophoresistechniques are used. Exemplified below is a commercially availableRP-HPLC column using a commercially prescribed RP-HPLC protocol. Thisfinal purification yields the two polypeptides in substantially pureform. "Substantially pure" means that a polypeptide contains less thanabout 5% by weight contaminants.

EXAMPLE

The following example is intended to illustrate the process forpurification as applied to a particular sample. It is not intended tolimit the invention.

A. Preparation of Demineralized Bone

Bovine metatarsal bone was obtained fresh from the slaughterhouse andtransported on dry ice. The bones were cleaned of marrow and non-bonetissues, broken fragments smaller than 1 cm diameter, and pulverized ina mill at 4° C. The pulverized bone was washed twice with 9.4 liters ofdouble distilled water per kg of bone for about 15 min each, and thenwashed overnight in 0.01N HCl at 4° C. Washed bone was defatted using3×3 volumes ethanol, followed by 3×3 volumes diethylether, each washedfor 20 min, and all at room temperature. The resulting defatted bonepowder was then demineralized in 0.5N HCl (25 l/kg defatted bone) at 4°C. The acid was decanted and the resulting DMB washed until the wash pHwas greater than 4, and the DMB dried on a suction filter.

B. Extraction of Noncollagenous Proteins

The DMB as prepared in paragraph A was extracted with 3.3 l of 4Mguanidine-HCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 6.8, 1mM PMSF, 10 mM NEM per kg for 16 hrs, the suspension suction filteredand the non-soluble fractions were combined and concentrated at least5-fold by ultrafiltration using an Amicon ultrafiltration (10K) unit,and the concentrate dialyzed against 6 changes of 35 volumes colddeionized water over a period of 4 days, and then lyophilized. All ofthe procedures of this paragraph were conducted at 4° C. except thelyophilization which was conducted under standard lyophilizationconditions.

C. Gel Filtration

The extract from paragraph B, redissolved in 4 M guanidine-HCl, wasfractionated on a Sephacryl S-200 column equilibrated in 4Mguanidine-HCl, 0.02% sodium azide, 10 mM EDTA, pH 6.8. Fractions wereassayed by their absorbance at 280 nm and chondrogenic activity by ELISAand the fractions were combined as shown in FIG. 1. Fraction F2 of FIG.1, constituting a low molecular weight (LMW, 10,000-40,000 daltons)protein fraction possessing the greatest activity was dialyzed against 6changes of 180 volumes of deionized water and lyophilized. Alloperations except lyophilization and dialysis (4° C.) were conducted atroom temperature.

D. Ion Exchange Chromatography

Fraction F2 from paragraph C was dissolved in 6M urea, 10 mM NaCl, 1 mMNEM, 50 mM sodium acetate, pH 4.8 and centrifuged at 10,000 rpm for 5min. The supernatant was fractionated on a CM52 (a commerciallyavailable CMC) column equilibrated in the same buffer. Bound proteinswere eluted from the column using a 10 mM to 400 mM NaCl gradient in thesame buffer, and a total volume of 350 ml at a flow rate of 27 ml/hr.Three major fractions, designated CM-1, CM-2, and CM-3 were collected asshown in FIG. 2. CM-2 and CM-3 eluted at approximately 150-250 mM NaCl.Each fraction was dialyzed against 6 changes of 110 volumes of deionizedwater for 4 days and lyophilized. All of the foregoing operations wereconducted at room temperature except dialysis (4° C.).

E. RP-HPLC

The lyophilized fractions CM-1 and combined lyophilized fractions CM-2and CM-3 from ¶D were each dissolved in 0.1% trifluoroacetic acid (TFA)and aliquots of the solutions loaded onto a Vydac C18 RP-HPLC column(4.6 mm ID×25 cm) and washed with 0.1% TFA for 5 min at 1 ml/min. Theeluting solvent was a 0%-60% acetonitrile gradient in 0.1% TFA at a rateof 2%/min.

FIG. 3 shows the chromatogram of the RP-HPLC of CM-1. Peak 3 containspredominantly 28 Kd chondrogenic/osteogenic co-factor protein and peak 6contains predominantly 36 Kd chondrogenic/osteogenic co-factor protein.Upon rechromatography using the same RP-HPLC system, the twopolypeptides were purified to give single bands on 15% SDS-PAGE(analysis also shown in FIG. 3). These protein co-factors are not partof the present invention and are the subject of a separate, commonlyowned U.S. patent application.

Two peaks were obtained from the RP-HPLC of combined CM-2 and CM-3--peakA at about 29.5 min and peak B at about 31.2 min. FIG. 4 shows theabsorbance and electrophoretic profiles (reduced and nonreduced) ofpeaks A and B. The proteins of these peaks were designated CIF-A andCIF-B, respectively.

The proteins were stored in 0.1% TFA/acetonitrile eluting solution at-20° C. until used.

F. Alternate Purification by Gel Electrophoresis

The combined lyophilized fractions CM-2 and CM-3 were fractionated byelectrophoresis on an acetic acid-urea gel using the general procedureof Paynim, S. and Chalkley, R., Arch Bioch Biophys (1969) 130:337-346.

G. Assay for In Vitro Chondrogenic Activity

The presence of the desired protein in fractions during purification wasconfirmed using an in vitro assay for the production ofcartilage-specific proteoglycans (PG), the identity of which wasconfirmed by ELISA. This assay is an agarose gel culture model usingmesenchymal cells isolated from rat fetal muscle. It assesses theability of the samples to induce the production of PG. The correlationbetween in vitro cartilage induction and in vivo bone formation has beenshown by Seyedin, S., et al. J Cell Biol (1983) 97:1950-1953.

The cell culture was prepared by removing muscle tissue aseptically fromthe upper limbs of nineteen-day-old Sprague Dawley rat fetuses, mincingthe tissue and culturing it in Eagle's Minimum Essential Medium (MEM)with 10% fetal bovine serum (FBS) and 50 units penicillin, 50 μgstreptomycin per ml. Cellular outgrowth usually reached confluencywithin one week, whereupon cells were trypsinized, split 1:2 and usedfor experimentation within the first three passages.

The cells were placed in agarose gel cultures either with control mediumor with samples to be tested. The procedure was basically that of Benya,et al, Cell (1982) 30:215. Briefly, the cell monolayers were harvestedby trypsinization, counted on a hemocytometer, and resuspended at twotimes the final cell concentration in the medium with or without theprotein fraction to be tested. The control medium was either Hams F-12,Dulbecco's Minimum Essential Medium (DMEM) or CMRL 1066 (Gibco) eachcontaining 10% FBS and antibiotics. The test protein fractions in 0.01NHCl were diluted directly to the desired concentration of test proteindiluted with an equal volume with 1% low melting agarose (Bio-Rad,#162-0017) in F-12, and 0.2 ml of the dilution was plated on 17 mm wellscoated with 0.15 ml of 1% high melting (Bio-Rad, #162-0100) agarose. Theresulting cultures were incubated at 37° C. for 5 min, chilled at 4° C.for 10 min, and then overlayed with 1 ml of the corresponding medium(control or test protein). The cells were then cultured in a humidifiedatmosphere of 5% CO₂, 95% air and fed every 3-4 days thereafter by acomplete change with control medium. After 7 days the cultures werefrozen and stored at -80° C. before assay.

The cultures were assayed by thawing at 4° C., homogenizing in 4Mguanidine-HCl with 50 nM Na acetate, 13 mM EDTA, 6 mM NEM, and 3 nM PMSFat pH 5.8, and extracting by shaking overnight at 4° C. The supernatantfraction from centrifugation at 25,000×g for 40 min at 4°C. was dialyzedovernight at 4° C. against 50 volumes 0.2 M NaCl, 50 mM Tris, pH 7.4.The supernatant was assayed for PG by ELISA as described by Renard, etal, Anal Biochem (1980) 104:205, and in U.S. Pat. No. 4,434,094.

Briefly, for the ELISA, antiserum to PG was raised in rabbits usingstandard techniques which showed no cross-reactivity with hyaluronicacid or PG extracted from rat bone. Purified PG (Seyedin, S., et al,supra) from Swarm rat chondrosarcoma tissue was used as standardantigen. The dialyzed samples were diluted 1:1 (v/v) inphosphate-buffered saline (PBS) with 0.05% Tween 20, 1 mg/ml bovineserum albumin (BSA), pH 7.2 for assay. Horseradish peroxidase conjugatedgoat anti-rabbit IgG (Tago) was the second antibody witho-phenylenediamine as substrate.

The results of the ELISA of the RP-HPLC purified CIF-A and CIF-B areshown in FIG. 5. As indicated there, the sensitivity of the assay iswithin 1 to 5 ng/ml of culture media. The results of the ELISAs on thegel slices of section F are shown in FIG. 6. These results arecomparable to the results on CIF-A and CIF-B (corresponding to gelslices 7 and 6) from the RP-HPLC.

H. Characterization of the Purified Proteins

CIF-A was shown to be a 25,800 dalton protein which on reduction,yielded a 14,800 dalton polypeptide by measurements of the mobilities ofthe proteins in a 15% Laemmli polyacrylamide gel in SDS (FIG. 4) asdescribed by Laemmli, U. K., et al, Nature (1970) 227:680. It is wellunderstood that molecular weights so determined are approximate andtheir values are dependent on the method used. The conformation of theprotein affects its mobility in this system, and, therefore, themolecular weights obtained will be similar, but no necessarily identicalwhen determined by other procedures. The presence of a single band inthe profile of the reduced protein indicates the protein is probably adimer composed of two polypeptide chains having substantially equivalentamine acid sequences (i.e., it is a homodimer). The discrepancy betweenthe measured weights of dimer and the individual chains is an artifactof the procedure.

CIF-A maintained its activity in the ELISA assay of paragraph G aboveeven after heating for 3 min at 100° C. in PBS, after treatment withcollagenase for 2 hrs at 37° C. in 0.1M Tris, pH 7.4, 5 mM CaCl₂, 0.02mM PMSF with a ratio of collagenase to protein of 400 units/mg protein,and after treatment with trypsin for 2 hrs at 37° C. in 50 mM Tris, pH7.4, 10 mM CaCl₂ with a ratio of trypsin to protein of 100 units/mg ofprotein. However, the protein lost activity after treatment for 1 hr atroom temperature in PBS containing 5 mM dithiothreitol (DTT), whichwould effect reduction of disulfide linkages. Similarly, SDS treatmentor fractionation on SDS-PAGE resulted in inactivation of the protein,presumably due to denaturation or complexing by the SDS. The partialamino acid composition of the CIF-A is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Amino Acid  Mols/100 Mols Recovered                                           ______________________________________                                        Asp         9.2                                                               Glu         9.2                                                               Ser         7.0                                                               His         2.7                                                               Gly         16.5                                                              Thr         2.7                                                               Arg         5.9                                                               Ala         6.6                                                               Tyr         3.2                                                               Met         0.0                                                               Val         7.5                                                               Phe         3.0                                                               Ile         3.9                                                               Leu         8.6                                                               Lys         13.9                                                              Pro         ND                                                                Cys         ND                                                                Trp         ND                                                                ______________________________________                                    

Amino acid sequence analysis of the protein of peak A by the ninhydrinmethod showed that is has the following single N-terminal sequence:##STR1## This N-terminal sequence is identical to that reported forhuman placenta-derived TGF-β.

CIF-B had a slightly different molecular weight (26,000) as measured bythe same procedure. This difference may be caused by the procedure.Accordingly, both proteins are considered to have a molecular weight ofapproximately 26,000 daltons as measured by SDS-PAGE. On reduction,CIF-B showed a single band at approximately 14,200 daltons indicatingthat it, too, is probably a homodimer. It has the amino acid compositionset forth in Table 2.

                  TABLE 2                                                         ______________________________________                                        Amino Acid  Mols/100 Mols Recovered                                           ______________________________________                                        Asp         12.0                                                              Glu         8.5                                                               Ser         10.6                                                              His         0.9                                                               Gly         22.0                                                              Thr         0.0                                                               Arg         4.3                                                               Ala         6.7                                                               Tyr         1.9                                                               Met         0.0                                                               Val         2.4                                                               Phe         3.0                                                               Ile         2.2                                                               Leu         8.2                                                               Lys         17.3                                                              Pro         ND                                                                Cys         ND                                                                Trp         ND                                                                ______________________________________                                    

Amino acid sequence analysis indicated CIF-B has a single N-terminalsequence as follows: ##STR2## Its other properties, as qualitativelyassessed, were similar to those set forth above for CIF-A.

I. Assay for TGF-β Activity

CIF-A and CIF-B were tested in the TGF-β bioassay. This assay determinesthe ability of a polypeptide to induce anchorage-independent growth innon-neoplastic normal rat kidney (NRK) fibroblasts by measuring theformation of colonies of cells in soft agar. The results of the assaysare shown in FIG. 7. As depicted, both proteins exhibit a cleardose-response in the assay and require the presence of an activatingagent (EGF) to be active. The levels of activity are comparable to thereported levels of activity of human platelet/human placenta/bovinekidney-derived TGF-β. As used herein the term "TGF-β activity" meansthat a material is active in the above-described assay.

J. In Vivo Cartilage Induction with Co-factors

1. Formulation

A collagenous carrier was prepared by mixing collagen in solution (CIS,1-3 mg protein/ml; sold by Collagen Corporation under the trademarkVITROGEN 100® with bone collagen powder (BCP, lyophilized solids frombone collagen) to give a minimal final concentration of 10% collagenfrom CIS. The 28 Kd or 36 Kd co-factors of ¶E were formulated with CIF-Aand CIF-B and the carrier. CIF-A and CIF-B were present in a weightratio of 2:1. The weight ratio of CIF to carrier was 1:1200, 1:4500,1:6000, 1:8000, or 1:20000. The formulations were stirred for 1-2 hr at4° C. and either directly lyophilized or dialyzed against water andlyophilized. Chondrogenic combinations of CIF-A/CIF-B and theseco-factors are not part of the present invention and are the subject ofa separate, commonly owned U.S. patent application. They are includedherein to illustrate the in vivo chondrogenic activity of CIF-A andCIF-B.

2. Biological Assays of Formulation of H

Implantation

The lyophilized formulations were rehydrated with two parts by weight ofcold sterile water and mixed to form a homogeneous paste. The rehydratedmaterial was formed into compact pellets (80-100 mg wet weight). Thepellets were implanted subcutaneously in the ventral thoracic region ofyoung male rats. Each rat received bilateral implants. The explants wererecovered at 14 days post implantation and evaluated histologically andbiochemically.

Histological Evaluation

Explants were fixed in 10% neutral formalin and paraffin embedded byroutine methods. Sections were subsequently stained with eitherhamatoxylin-eosin or with Gomori trichome.

Biochemical Assays

The explants were split in half, their wet weight determined, and frozenat -80° C. until processed. The samples were first extracted and assayedfor alkaline phosphate activity and subsequently extracted and assayedfor cartilage-specific proteoglycans.

Results

Histological evaluations showed that the implants induced cartilagegrowth at the implant site. Evaluations of comparison implantscontaining only CIF or only co-factor showed no cartilage development.Similarly, in the biochemical assays the implants showed high cartilagePG production whereas the comparison implants did not. Comparisonimplants containing only CIF in which the CIF to carrier weight ratiowas greater than about 1:6000 developed a dense collagenous connectivetissue matrix throughout the implant. This ability to promote connectivetissue deposition does not appear to require association with anyco-factor or activating agent that is required for chondrogenic activityof TGF-β activity.

The proteins of the invention are useful in combination with co-factorssuch as the above described 28 Kd and 36 Kd proteins for inducingcartilage/bone formation for repairing, replacing or augmentingcartilage/bone tissue in animals including humans.Chondrogenically/osteogenically effective amounts of the proteins willnormally be formulated with pharmacologically and physiologicallyacceptable fluid or solid carriers for implantation. The weight ratio ofCIF to carrier will typically be in the range of 1:1000 to 1:20000. Theimplants may be placed at a predetermined site in the patient byconventional surgical techniques including injection when formulated asan injectable. When used to promote connective tissue deposition, theCIF is combined in effective amounts with carriers conventionally usedfor implantation. When collagen carrier is used, it appears from thetests reported above that CIF:carrier weight ratios greater than about1:6000 are required. The amount of CIF may vary when it is combined withother carriers.

The CIFs of the invention may also be used in the same manner as TGF-βto promote (provoke and sustain) non-species specific cellularproliferation. In such application the proteins are normally combined inapproximately stoichiometric proportions with a TGF-β activating agentsuch as an EGF or a TGF-β. Clinical applications of the cellproliferation activity of these compositions include topicaladministration for burn or wound healing or tissue repair. In such usesthe proteins and activating agent will be formulated in amountssufficient to induce soft tissue cell proliferation withpharmaceutically acceptable carriers that are adapted for the particularmode of administration. Topical dosage forms will typically beformulated as sprays, gels, ointments, or salves. Implants will beformulated as injectables. Systemic dosage forms may be formulated forenteral administration (e.g., liquids, pills, tablets) or for parenteralinjection. The dosages used in such applications cannot be specifiedbecause of the nature of all proliferation activity and the variabilityin wounds and other traumata.

The CIFs may also be useful for treating bone deficiencies, such asosteoporosis and osteopetrosis, systemically. For such treatment theCIFs will be formulated in therapeutically effective amounts withinjectable carriers and administered parenterally to the patient.

The ability of the CIFs to withstand treatment with trypsin withoutlosing activity may make it possible to isolate them from demineralizedbone powder by means of enzymatic digestion. In such a process thedemineralized bone powder is digested with an aqueous solution oftrypsin and/or other proteases that do not degrade the proteins ofinterest under conditions at which such enzymes are active. Thistreatment digests the majority of other protein components in thepowder. The proteins of interest may be purified from the resultingdigest using one or more of the fractionation techniques described above(gel filtration, ion exchange chromatography, RP-HPLC or nondenaturinggel electrophoresis). Depending upon the extent to which the proteins ofinterest are released from the bone matrix and not complexed with othermaterials, use of solubilizing agents may be avoided. In this regard thepure CIFs are substantially soluble in water.

We claim: . .1. A method of promoting proliferation of cells in ananimal comprising Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp-..!...5. A method for treating osteoporosis or osteopetrosis, whichcomprises:systemically administering an effective amount of acomposition comprising a TGF-beta activating agent, a pharmaceuticallyacceptable excipient, and a polypeptide that (a) is found in mammalianbone, (b) is active in the TGF-beta assay, (c) is a co-factor forinducing cartilage formation in vivo, (d) is a dimer having anapproximate molecular weight of 26,000 daltons as determined bySDS-PAGE, and (e) is substantially pure, wherein said polypeptide andactivating agent are present in amounts sufficient to promote bonegrowth..!.. .6. The method of claim 5 wherein each chain of said dimerhas the following N-terminal sequence:Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp-..!...7. The method of claim 3 wherein each chain of said dimer has thefollowing N-terminal sequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp..!...8. The method of claim 5 wherein each chain of said dimer has thefollowing N-terminal sequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp..!..Iadd.9. A method forinducing cartilage and bone formation for repairing, replacing, and/oraugmenting cartilage and bone tissue in animals, which method comprisesadministering to an animal in need of bone repair, replacement, oraugmentation a composition comprising a TGF-beta activating agent, apharmaceutically acceptable excipient, and a polypeptide that (a) isfound in mammalian bone, (b) is active in the TGF-beta assay, (c) is aco-factor for inducing cartilage formation in vivo, (d) is a dimerhaving an approximate molecular weight of 26,000 daltons as determinedby SDS-PAGE, and (e) is substantially pure, wherein said polypeptide andactivating agent are present in amounts sufficient to promote bonegrowth..Iaddend..Iadd.10. The method of claim 9 wherein each chain ofsaid dimer has the following N-terminalsequence:Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp(TGF-β1)..Iaddend..Iadd.11. The method of claim 9 wherein each chain ofsaid dimer has the following N-terminal sequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp(TGFβ-2)..Iaddend..Iadd. . A method for treating osteoporosis orosteopetrosis, which comprises systemically administering to anindividual in need of such treatment an effective amount of acomposition comprising a TGF-beta activating agent, a pharmaceuticallyacceptable excipient, and a polypeptide that (a) is found in mammalianbone, (b) is active in the TGF-beta assay, (c) is a co-factor forinducing cartilage formation in vivo, (d) is a dimer having anapproximate molecular weight of 26,000 daltons as determined bySDS-PAGE, and (e) is substantially pure, wherein said polypeptide andactivating agent are present in amounts sufficient to promote bonegrowth..Iaddend..Iadd.13. The method of claim 12 wherein each chain ofsaid dimer has the following N-terminalsequence:Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp(TGFβ-1)..Iaddend..Iadd.14. The method of claim 12 wherein each chain ofsaid dimer has the following N-terminal sequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp(TGFβ-2)..Iaddend..Iadd.15. A method for inducing cartilage and boneformation for repairing, replacing, and/or augmenting cartilage and bonetissue in animals, which method comprises administering to an animal inneed of bone repair, replacement, or augmentation a compositioncomprising a pharmaceutically acceptable excipient and a polypeptidethat (a) is found in mammalian bone, (b) is active in the TGF-β assay,(c) is a co-factor for inducing cartilage formation in vivo, (d) is adimer having an approximate molecular weight of 26,000 daltons asdetermined by SDS-PAGE, and (e) is substantially pure, wherein saidpolypeptide is present in an amount sufficient to promote bonegrowth..Iaddend..Iadd.16. The method of claim 15 wherein each chain ofsaid dimer has the following N-terminalsequence:Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp(TGFβ-1)..Iaddend..Iadd.17. The method of claim 15 wherein each chain ofsaid dimer has the following N-terminal sequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp(TGFβ-2)..Iaddend..Iadd.18. A method for treating osteoporosis orosteopetrosis, which comprises systemically administering to anindividual in need of such treatment an effective amount of acomposition comprising a pharmaceutically acceptable excipient and apolypeptide that (a) is found in mammalian bone, (b) is active in theTGF-β assay, (c) is a co-factor for inducing cartilage formation invivo, (d) is a dimer having an approximate molecular weight of 26,000daltons as determined by SDS-PAGE, and (e) is substantially pure,wherein said polypeptide is present in an amount sufficient to promotebone growth..Iaddend..Iadd.19. The method of claim 18 wherein each chainof said dimer has the following N-terminal sequence:Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser-(Ser)-Thr-Glu-Lys-Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp(TGFβ-1)..Iaddend..Iadd.0. The method of claim 18 wherein each chain ofsaid dimer has the following N-terminalsequence:Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-Cys-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly-Trp(TGFβ-2)..Iaddend.